The goal of this proposal is to study the mechanism o interaction o a new potentially attractive drug, Peptide Nucleic Acid (PNA), with double- stranded (ds) DNA. The unique property of the drug will be investigated, which consists in exceptionally strong and highly sequence-specific binding to dsDNA via strand displacement reaction. To study this totally new mode of targeting dsDNA, special assays will be developed. The electrophoretic mobility shift assay will be based on different mobility in the course of polyacrylamide gel electrophoresis of a dsDNA fragment and its complex with PNA. The nuclease cutting assay will be based on the ability of single strand-specific nucleases to cut dsDNA at the sites where PNA binds to dsDNA. The electron-microscopy assay will use biotinylated PNA and streptavidin as a marker of the sites of the PNA association with dsDNA. Scanning force microscopy will be used to study the fine structure of PNA/dsDNA complexes. These methods will be used for quantitative studies of the kinetics of the strand displacement reaction for PNA oligomers of different length and sequence as a function of ambient conditions. Bis-PNA, consisting of two PNA oligomers connected by a flexible linker, PNAs with lysine "tails" of different length, intercalator-tagged PNAs, DNA-PNA conjugates, DNA-bis- PNA conjugates, PNA containing specially designed base analogs and many other PNA derivatives will also be studied. The accumulated data will be used to formulate the detailed theoretical model of the strand- displacement reaction. The key point in the model will be the PNA~NA triplex between purine DNA strand and two homopyrimidine PNA molecules (or two halves of bis-PNA). Dissociation (melting) kinetics of DNA~PNA complexes will be studied for various model systems, such as DNA/PNA duplexes and triplexes, to determine fundamental kinetic parameters. Extensive theoretical modeling of the various pathways of the process of the strand displacement will be performed by the Monte Carlo method. Numerous consequences of the theoretical modeling will be tested and the model will be adjusted to satisfy the whole body of experimental data. The validity of the model not only for homopyrimidine PNA/dsDNA complexes but also for homopurine PNA/dsDNA complexes will be also tested. The correlation between the sequence specificity of PNA/dsDNA interaction and the mechanism of the reaction will be established. The major result of the project from the basic viewpoint will be understanding of an entirely new mode of DNA targeting. The pivotal question will be answered about the factors determining both strong and sequence specific binding of PNA with DNA. In practical terms, the major outcome of the project will consist in finding ways to improve efficiency and sequence specificity of PNA-like drugs. The methods elaborated will make it possible to screen various PNA analogs with respect to their ability to target specific sites at genomes. In particular, the polypurine tract in HTV-I genome will be targeted in the form of duplex DNA and in the form of a DNA/RNA heteroduplex.